The detection, analysis, transcription and amplification of nucleic acids are the most important procedures in modern molecular biology. The application of such procedures for RNA analysis is especially important in the investigation of gene expression, diagnosis of infectious agents or genetic diseases, the generation of cDNA and analysis of retroviruses, to name but a few applications. The reverse transcription of RNA, followed by polymerase chain reaction amplification, commonly referred to as RT-PCR, has become widely used for the detection and quantification of RNA.
The RT-PCR procedure involves two separate molecular syntheses: First, the synthesis of cDNA from an RNA template; and second, the replication of the newly synthesized cDNA through PCR amplification. RT-PCR may be performed under three general protocols:                1) Uncoupled RT-PCR, also referred to as two-step RT-PCR.        2) Single enzyme coupled RT-PCR, also referred to as one-step RT-PCR or continuous RT-PCR, in which a single polymerase is used for both the cDNA generation from RNA as well as subsequent DNA amplification.        3) Two (or more) enzyme coupled RT-PCR, in which at least two separate polymerases are used for initial cDNA synthesis and subsequent replication. This is sometimes also referred to as one-step RT-PCR or, alternatively, one-tube RT-PCR.        
In uncoupled RT-PCR, reverse transcription is performed as an independent step using buffer and reaction conditions optimal for reverse transcriptase activity. Following cDNA synthesis, an aliquot of the RT reaction product is used as template for PCR amplification with a thermostable DNA polymerase, such as Taq DNA Polymerase, under conditions optimal for PCR amplification.
In coupled RT-PCR, reverse transcription and PCR amplification are combined into a single reaction mixture. Single enzyme RT-PCR utilizes the reverse transcriptase activity of certain DNA polymerases, such as Tth DNA polymerase, whereas two-enzyme RT-PCR typically uses a retroviral or bacterial reverse transcriptase (e.g. AMV-RT, MMLV-RT, HIV-RT, EIAV-RT, RAV2-RT, Carboxydothermus hydrogenoformans DNA polymerase or a mutant, variant or derivative thereof), and a thermostable DNA polymerase (e.g. Taq, Tbr, Tih, Tfi, Tfl, Pfu, Pwo, Kod, VENT™, DEEPVENT™, Tma, Tne, Bst, Pho, Sac, Sso, ES4 and others or a mutant, variant or derivative thereof).
Coupled RT-PCR provides numerous advantages over uncoupled RT-PCR. Coupled RT-PCR requires less handling of the reaction mixture reagents and nucleic acid products than uncoupled RT-PCR (e.g., opening of the reaction tube for component or enzyme addition in between the two reaction steps), and is therefore less labor-intensive, and time-consuming, and has reduced risk of contamination. Furthermore, coupled RT-PCR also requires less sample, making it especially suitable for applications where the sample amounts are limited (e.g., with FFPE, biopsy, environmental samples).
Although single-enzyme-coupled RT-PCR is easy to perform, this system is expensive to perform, however, due to the amount of DNA polymerase required. In addition, the single enzyme coupled RT-PCR method has been found to be less sensitive than uncoupled RT-PCR, and limited to polymerizing nucleic acids of less than one kilobase pair in length. Coupled RT-PCR systems with two or more enzymes generally show increased sensitivity over the single enzyme system, even when coupled in a single reaction mixture. This effect has been attributed to the higher efficiency of reverse transcriptase in comparison to the reverse transcriptase activity of DNA polymerases (Sellner and Turbett, BioTechniques 25(2):230-234 (1998)).
Although the two-enzyme coupled RT-PCR system is more sensitive than the single-enzyme system, reverse transcriptase has been found to interfere directly with DNA polymerase during the replication of the cDNA, thus reducing the sensitivity and efficiency of this technique (Sellner et al., J. Viol. Methods 40:255-264 (1992)). A variety of solutions to overcome the inhibitory activity of reverse transcriptase on DNA polymerase have been tried, including: increasing the amount of template RNA, increasing the ratio of DNA polymerase to reverse transcriptase, adding modifier reagents that may reduce the inhibitory effect of reverse transcriptase on DNA polymerase (e.g., non homologous tRNA, T4 gene 32 protein, sulphur or acetate-containing molecules), and heat-inactivation of the reverse transcriptase before the addition of DNA polymerase.
Sellner et al. (Nucleic Acids Research 20(7):1487-1490) describe that the detection of viral RNA by polymerase chain reaction requires the prior reverse transcription of the viral RNA. In order to minimize the number of manual manipulations required for processing large numbers of samples, Sellner et al. attempted to design a system whereby all the reagents required for both reverse transcription and amplification can be added to one tube and a single, non-interrupted thermal cycling program can be performed. Whilst attempting to set up such a one-tube system with Taq polymerase and avian myoblastis virus, they noticed a substantial decrease in the sensitivity of detection of viral RNA. They found out a direct interference of reverse transcriptase with Taq polymerase.
All of these modified RT-PCR methods have significant drawbacks, however. Increasing the amount of template RNA is not possible in cases where only limited amounts of sample are available. Individual optimization of the ratio of reverse transcriptase to DNA polymerase is not practicable for ready-to-use reagent kits for one-step RT-PCR. The net effect of currently proposed modifier reagents to relive reverse transcriptase inhibition of DNA polymerization is controversial and in dispute: positive effects due to these reagents are highly dependent on RNA template amounts, RNA composition, or may require specific reverse transcriptase-DNA polymerase combinations (Chandler et al., Appl. and Environm Microbiol. 64(2):669-677 (1998)). Finally, heat inactivation of the reverse transcriptase before the addition of the DNA polymerase negates the advantages of the coupled RT-PCR and carries all the disadvantages of uncoupled RT-PCR systems discussed earlier. Even if a reverse transcriptase is heat inactivated, it still may confer an inhibitory effect on PCR, likely due to binding of heat-inactivated reverse transcriptase to the cDNA template.
Some improvements to reduce the inhibitory effect of reverse transcriptase on the activity of the polymerase have been made.
In US 2009/0137008 A1, Gong and Wang describe the reduction of the inhibitory effect of reverse transcriptase on DNA polymerase by proteins that bind dsDNA in a non-specific way such as Sso7d, Sac7d, Sac7e or Sso7e and by sulfonic-acid and by sulfonic acid salts.
In EP 1050587 B1, Missel et al. describe the reduction of the inhibitory effect of reverse transcriptase on DNA polymerase by homopolymeric nucleic acids.
Although the methods described by Gong and Wang and Missel et al., respectively, successfully have shown a significant reduction of the inhibitory effect of reverse transcriptase, a further improved specificity and sensitivity of RT-PCR by a more effective reduction of the inhibitory effect of reverse transcriptase is still a need in the art.
Because of the importance of RT-PCR applications, a coupled RT-PCR system, in the form of a generalized ready-to-use composition, which exhibits high specificity and sensitivity, requires a small amount of initial sample, reduces the amount of practitioner manipulation, minimizes the risks of contamination, minimizes the expense of reagents, is not restricted to the use of specific reaction buffers, and maximizes the amount of nucleic acid end product is needed in the art.